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PubMed2003

High-pressure FTIR study of the stability of horseradish peroxidase. Effect of heme substitution, ligand binding, Ca++ removal, and reduction of the disulfide bonds.

The pressure stability of horseradish peroxidase isoenzyme C and the identification of possible stabilizing factors are presented. The effect of heme substitution, removal of Ca(2+), binding of a small substrate molecule (benzohydroxamic acid), and reduction of the disulfide bonds on the pressure stability were investigated by FTIR spectroscopy. HRP was found to be extremely stable under high pressure with an unfolding midpoint of 12.0 +/- 0.1 kbar. While substitution of the heme for metal-free mesoporphyrin did not change the unfolding pressure, Ca(2+) removal and substrate binding reduced the midpoint of the unfolding by 2.0 and 1.2 kbar, respectively. The apoprotein showed a transition as high as 10.4 kbar. However, the amount of folded structure present at the atmospheric pressure was considerably lower than that in all the other forms of HRP. Reduction of the disulfide bonds led to the least pressure stable form, with an unfolding midpoint at 9.5 kbar. This, however, is still well above the average pressure stability of proteins. The high-pressure stability and the analysis of the pressure-induced spectral changes indicate that the protein has a rigid core, which is responsible for the high stability, while there are regions with less stability and more conformational mobility.

Amides↗
PubMed2003

Remarkable stabilization of neutrophil NADPH oxidase using RacQ61L and a p67phox-p47phox fusion protein.

Activation of the phagocyte NADPH oxidase occurs via assembly of cytosolic p47(phox), p67(phox), and Rac with the membrane-bound flavocytochrome b(558). Recently, we have found that p67(phox)-(1-210) (p67N) fused with p47(phox)-(1-286) (p47N) or with Rac efficiently stabilizes the oxidase in a cell-free reconstitution system. In an attempt to further stabilize the oxidase, we herein used a constitutively active Rac, RacQ61L, and examined its effect on the oxidase stability. The half-life (t(1/2)) of the activity reconstituted with wild-type Rac was 12 min at 37 degrees C, which was extended 6-fold by RacQ61L. Also, the stability of the oxidase without p47(phox) increased 8-fold using RacQ61L. RacQ61L had a higher affinity for the complex than wild-type Rac and increased the affinity of p67N for the complex. Far-western blotting showed an enhanced binding between RacQ61L and p67N. The oxidase was stabilized by nanomolar FAD, and RacQ61L lowered the FAD concentration required. The combination of RacQ61L and a fusion protein consisting of p67N and p47N produced an extremely stable enzyme (t(1/2) = 184 min at 37 degrees C). The effectiveness of RacQ61L and fusion proteins on stabilization was in the following order: p67N-Rac < p67N + RacQ61L < or = p67N-RacQ61L << p67N-p47N + RacQ61L. These results indicate that a tightly bound ternary complex of p67(phox), Rac, and p47(phox) is very effective in maintaining the oxidase and confirm that the longevity of the activated state requires continuous association of these components. This simple and efficient method of stabilization may provide a useful tool to elucidate the nature of the activated oxidase.

Enzyme Activation↗
PubMed2006

Completely buried, non-ion-paired glutamic acid contributes favorably to the conformational stability of pyrrolidone carboxyl peptidases from hyperthermophiles.

Pyrrolidone carboxyl peptidases (PCPs) from hyperthermophiles have a structurally conserved and completely buried Glu192 in the hydrophobic core; in contrast, the corresponding residue in the mesophile protein is a hydrophobic residue, Ile. Does the buried ionizable residue contribute to stabilization or destabilization of hyperthermophile PCPs? To elucidate the role of the buried glutamic acid in stabilizing PCP from hyperthermophiles, we constructed five Glu192 mutants of PCP-0SH (C142S/C188S, Cys-free double mutant of PCP) from Pyrococcus furiosus and examined their thermal and pH-induced unfolding and crystal structures and compared them with those of PCP-0SH. The stabilities of apolar (E192A/I/V) and polar (E192D/Q) mutants were less than PCP-0SH at acidic pH values. In the alkaline region, the mutant proteins, except for E192D, were more stable than PCP-0SH. The thermal stability data and theoretical calculations indicated an apparent pKa value > or = 7.3 for Glu192. Present results confirmed that the protonated Glu192 in PCP-0SH forms strong hydrogen bonds with the carbonyl oxygen and peptide nitrogen of Pro168. New intermolecular hydrogen bonds in the E --> A/D mutants were formed by a water molecule introduced into the cavity created around position 192, whereas the hydrogen bonds disappeared in the E --> I/V mutants. Structure-based empirical stability of mutant proteins was in good agreement with the experimental results. The results indicated that (1) completely buried Glu192 contributes to the stabilization of PCP-0SH because of the formation of strong intramolecular hydrogen bonds and (2) the hydrogen bonds by the nonionized and buried Glu can contribute more than the burial of hydrophobic groups to the conformational stability of proteins.

Calorimetry, Differential Scanning↗
PubMed1996

Contributions of the ionizable amino acids to the stability of staphylococcal nuclease.

To quantitate the contributions of the ionizable amino acids to the stability of the native state of staphylococcal nuclease, each of the 23 lysines, 5 arginines, 4 histidines, 12 glutamic acids, and 8 aspartic acids was substituted with both alanine and glycine. This collection of 104 mutant proteins was analyzed by guanidine hydrochloride (GuHCl) denaturation, using intrinsic tryptophan fluorescence to quantitate the equilibrium between native and denatured states. From the analysis of these data, each mutant protein's stability in the absence of denaturant (delta GH2O) and sensitivity to changes in denaturant concentration [mGuHCl = d(delta G)/d[GuHCl]] were obtained. Several general trends in these values suggest that electrostatic interactions make only a minor contribution to the net stability of this protein. For the residue pairs that form ten salt bridges and ten charged hydrogen bonds between side chains, no correlation was observed between the stability losses (delta delta G) accompanying alanine substitution of each member of the pair. Little or no significant correlation was found between the magnitude of the loss in stability and the local electrostatic potential calculated from the three-dimensional structure by numerical and model dependent solutions of the linearized Poisson-Boltzmann equation. The structural parameters which correlated most strongly with stability loss are measures of the extent of burial of the residue in the native structure, as was previously observed for alanine and glycine substitutions of large hydrophobic residues [Shortle et al. (1990) Biochemistry 29, 8033] and of the polar, uncharged residues [Green et al. (1992) Biochemistry 31, 5717]. These results suggest that the ionizable amino acids contribute to stability predominantly through packing and bonding interactions that do not depend on their electrostatic charge.

Amino Acids↗
PubMed1996

Relationship between stability and function for isolated domains of troponin C.

Results of spectroscopic thermal and chemical denaturation studies and calcium binding studies are presented for a series of five recombinant chicken troponin C fragments. They were designed to assess the effects of domain isolation, N-helix, and D/E linker helix on stability and calcium affinity. Four of the fragments include the N-terminal regulatory domain and one the C-terminal domain. For the regulatory domain, deletion of the N-helix or the D/E linker decreases the stability of the apo form as measured by delta GN-->U,25. Separation of the domains also decreases the stability. Differences in values of delta GN-->U,25 derived from urea and guanidine hydrochloride studies allowed an estimation of the electrostatic component of the free energy of unfolding. Our measurements provide the first quantitative estimate of the stability for the apo-C-domain (delta GN-->U,25 = -1.8 kcal/mol) which was obtained using the interaction free energy formalism of Schellman. There is an inverse correlation between calcium affinity, binding cooperativity, and stability for all of these homologously structured fragments. The calcium affinity and cooperativity are highest for the unstructured C-domain and lowest for the N-domain which has the highest stability. In view of the direct effects on the folding stability of the apo-N-domain, the N-helix and the bilobed domain organization of TnC are necessarily involved in the fine-tuning of the affinity and cooperativity of calcium binding. Though not directly involved in calcium coordination, these structural features are important for signal transmission by troponin C in the troponin complex.

Animals↗
PubMed1998

Context-dependent nature of destabilizing mutations on the stability of FKBP12.

The context-dependent nature in which mutations affect protein stability was investigated using the FK506-binding protein, FKBP12. Thirty-four mutations were made at sites throughout the protein, including residues located in the hydrophobic core, the beta-sheet, and the solvent-exposed face of the alpha-helix. Urea-induced denaturation experiments were used to measure the change in stability of the mutants relative to that of the wild type (Delta DeltaGU-F). The results clearly show that the extent of destabilization, or stabilization, is highly context-dependent. Correlations were sought in order to link Delta DeltaGU-F to various structural parameters. The strongest correlation found was between Delta DeltaGU-F and N, the number of methyl(ene) groups within a 6 A radius of the group(s) deleted. For mutations of buried hydrophobic residues, a correlation coefficient of 0.73 (n = 16,where n is the number of points) was obtained. This increased to 0.81 (n = 24) on inclusion of mutations of partially buried hydrophobic residues. These data could be superimposed on data obtained for other proteins for which similarly detailed studies have been performed. Thus, the contribution to stability from hydrophobic side chains, independent of the extent to which a side chain is buried, can be estimated quantitatively using N. This correlation appears to be a general feature of all globular proteins. The effect on stability of mutating polar and charged residues in the alpha-helix and beta-sheet was also found to be highly context-dependent. Previous experimental and statistical studies have shown that specific side chains can stabilize the N-caps of alpha-helices in proteins. Substitutions of Ile56 to Thr and Asp at the N-cap of the alpha-helix of FKBP12, however, were found to be highly destabilizing. Thus, the intrinsic propensities of an amino acid for a particular element of secondary structure can easily be outweighed by tertiary packing factors. This study highlights the importance of packing density in determining the contribution of a residue to protein stability. This is the most important factor that should be taken into consideration in protein design.

Arginine↗
PubMed1999

Comparison of local and global stability of an analogue of a disulfide-folding intermediate with those of the wild-type protein in bovine pancreatic ribonuclease A: identification of specific regions of stable structure along the oxidative folding pathway.

We have identified specific regions of the polypeptide chain of bovine pancreatic ribonuclease A (RNase A) that are critical for stabilizing the oxidative folding intermediate des-[40-95] (with three native disulfide bonds but lacking the fourth native Cys40-Cys95 disulfide bond) in an ensemble of largely disordered three-disulfide precursors (3S if des-[40-95]). A stable analogue of des-[40-95], viz., [C40A, C95A] RNase A, which contains three out of four native disulfide pairings, was previously found to have a three-dimensional structure very similar to that of the wild-type protein. However, it is determined here from GdnHCl denaturation experiments to have significantly reduced global stability, i.e., = 4.5 kcal /mol at 20 degrees C and pH 4.6. The local stability of [C40A, C95A] RNase A was also examined using site-specific amide (2)H/(1)H exchange measurements at pD 5.0 to determine the individual unfolding free energy of specific residues under both strongly native (12 degrees C) and more destabilizing (20 degrees C) conditions. Comparison of the relative stabilities at specific amide sites of [C40A, C95A] RNase A at both temperatures with the corresponding values for the wild-type protein at 35 degrees C corroborates previous experimental evidence that unidentified intramolecular contacts in the vicinity of the preferentially formed native one-disulfide (C65-C72) loop are crucial for stabilizing early folding intermediates, leading to des-[40-95]. Moreover, values of for residues at or near the third alpha-helix, and in part of the second beta-sheet of [C40A, C95A] RNase A, indicate that these two regions of regular backbone structure contribute to stabilizing the global chain fold of the des-[40-95] disulfide-folding intermediate in the wild-type protein. More significantly, we have identified numerous specific residues in the first alpha-helix and the first beta-sheet of the protein that are stabilized in the final step of the major oxidative regeneration pathway of RNase A (des-[40-95] --> N).

Amides↗
PubMed2001

Aminoglycoside-nucleic acid interactions: remarkable stabilization of DNA and RNA triple helices by neomycin.

The stabilization of poly(dA).2poly(dT) triplex, a 22-base DNA triplex, and poly(rA).2poly(rU) triple helix by neomycin is reported. The melting temperatures, the association and dissociation kinetic parameters, and activation energies (E(on) and E(off)) for the poly(dA).2poly(dT) triplex in the presence of aminoglycosides and other triplex binding ligands were determined by UV thermal analysis. Our results indicate that: (i) neomycin stabilizes DNA triple helices, and the double helical structures composed of poly(dA).poly(dT) are virtually unaffected. (ii) Neomycin is the most active and triplex-selective stabilization agent among all aminoglycosides, previously studied minor groove binders, and polycations. Its selectivity (DeltaT(m3-->2) vs DeltaT(m2)(-->)(1)) exceeds most intercalating drugs that bind to triple helices. (iii) Neomycin selectively stabilizes DeltaT(m3)(-->)(2) for a mixed 22-base DNA triplex containing C and T bases in the pyrimidine strand. (iv) The rate constants of formation of triplex (k(on)) are significantly enhanced upon increasing molar ratios of neomycin, making triplex association rates closer to duplex association rates. (v) E(on) values become more negative upon increasing concentration of aminoglycosides (paromomycin and neomycin). E(off) values do not show any change for most aminoglycosides except neomycin. (vi) Aminoglycosides can effectively stabilize RNA [poly(rA).2poly(rU)] triplex, with neomycin[being one of the most active ligands discovered to date (second only to ellipticine). (vii) The stabilization effect of aminoglycosides on triple helices is parallel to their toxic behavior, suggesting a possible role of intramolecular triple helix (H-DNA) stabilization by the aminoglycosides.

Aminoglycosides↗
PubMed2005

Effects of the combination of hydrophobic polypeptides, iso-alpha acids, and malto-oligosaccharides on beer foam stability.

The influence of hydrophobic polypeptides concentrated in beer foam, together with the composition of iso-alpha acids and the content of malto-oligosaccharides in beer on foam stability, has been investigated. The objective was to find out whether a shortage of one of these positive contributors to foam stability could be compensated for by an increased presence of another or whether optimum levels of each contributor is necessary. For that purpose, an image analysis method to evaluate beer foam quality was developed. The foam collapse time was the parameter chosen to group beers according to their foam stability. Profiles of hydrophobic polypeptides that concentrate in beer foam, iso-alpha acids, and malto-oligosaccharides of 14 beer brands were acquired by high-performance liquid chromatography. Principal component analysis (PCA) was performed to show the relationship between beer brands and its composition. Beers that contained propylene glycol alginate as a foam enhancer showed high foam stability except for one beer, which had a low content of hydrophobic polypeptides, thereby highlighting the requirement of threshold levels of hydrophobic polypeptides to obtain stable foam. The data of samples that were devoid of a foam additive were subjected to a discriminant statistical analysis. Foam stability declined in proportion to decreases in hydrophobic polypeptides and to a lesser extent to decreases in iso-alpha-acid contents. Apparently, the content of malto-oligosaccharides were found to have no major influence on foam stability. The model of discriminate analysis was found to explain 100% of the variance in data with 85.2% success in classifying all samples according to the model, suggesting that foam stability is mainly governed by the beer constituents evaluated in this study.

Acids↗
PubMed1996

Indium (III) and gallium (III) complexes of bis(aminoethanethiol) ligands with different denticities: stabilities, molecular modeling, and in vivo behavior.

Complexes of Ga(III) and In(III) radionuclides are widely used in diagnostic imaging. In this study, the following ligands of denticities 4, 5, and 6 respectively were prepared: N,N'-bis-(2,2-dimethyl-2-mercaptoethyl) ethylenediamine (4SS), 1-carboxy-N-N'-bis(2,2-dimethyl-2- mercaptoethyl)ethylenediamine (5SS), and N,N'-bis(2,2- dimethyl-2-mercaptoethyl)ethylenediamine-N,N'-diacetic acid (6SS). Syntheses of the two new ligands, 5SS and 6SS, are described. Equilibrium constants for their In(III) and Ga(III) complexes were determined by both direct and ligand-competitive potentiometric methods. The formation constant (KML = [ML]/[M][L]) of In(III)--6SS in 0.100 M KNO3 at 25.0 degrees C is 10(39.8), and its pM at physiological pH (7.4 with 100% excess of the ligand) is 30.9. These values are higher than those of any other previous reported ligand for In(III). The stability constants of the complexes of 4SS, 5SS, 6SS, and the analogous ligand EDDASS, N,N'-bis(2-mercaptoethyl) ethylenediamine-N,N'-diacetic acid, which does not contain gem-dimethyl groups, are compared. The thermodynamic stabilities of the In(III) complexes of all ligands except 6SS are greater than those of the corresponding Ga(III) complexes. The presence of the geminal dimethyl groups in 6SS increased the stability of the Ga(III) and In(III) complexes over those of EDDASS. The effects of the gem-dimethyl groups on complex stabilities are explained by molecular modeling. The serum stabilities and biodistributions out to 1 h postinjection of 67/68Ga and 111In chelates of 4SS, 5SS, and 6SS were measured and compared with those of EDDASS. The 67/68Ga- and 111In-ligand complexes with more donor atoms showed were more stable in serum, both in vitro and in vivo. The biodistributions of the 67/68Ga- and 111In-ligand complexes exhibited distinct trends. None of the 67/68Ga- and 111In-chelates demonstrated significant heart or brain uptake. The majority of uptake for all compounds was in the liver and kidney. The degree of clearance through the liver corresponded to the thermodynamic stability of the complex. Correlations between in vivo behavior, molecular modeling data, and thermodynamic stability of the complexes are discussed.

Animals↗
PubMed1996

Evaluation of the stability and animal biodistribution of gadolinium (III) benzylamine-derivatized diethylenetriaminepentaacetic acid.

The need for a readily available Gd(III) bifunctional chelate for protein conjugation has led to the development of LDTPA (N,N-bis[2-[N',N'-bis(carboxymethyl)amino]- ethyl]-4-amino-L-phenyl-alanine). The benzylamine group is readily converted to the isothiocyanato group (SCN-LDTPA) by treatment of the lithium salt of LDTPA with thiophosgene. SCN-LDTPA was successfully conjugated to three proteins, BSA (bovine serum albumin), mannose BSA, and galactose BSA. All protein conjugates were labeled with 111In3+ or 153Gd3+. Competition of Gd-LDTPA with DTPA (diethylenetriaminepentaacetic acid) resulted in a log stability constant of 21.2. The thermodynamic stability constant of Gd-LDTPA was also measured. The log Gd(III) stability constant (log K) is 21.99, and the log protonation constants (pKa's) are 10.16, 8.92, 5.35, 3.93, 2.71, and 1.89. Comparison of the thermodynamic stability constants for Gd(LDTPA)2- with other DTPA derivatives indicates that the stability of Gd(LDTPA)2- is similar to Gd(DTPA)2- (log K = 22.4), and higher than DTPA derivatives with one or more carboxylate arm(s) functionalized. The biodistribution of 153Gd-LDTPA-protein conjugates is consistent with the in vitro stability measurements. By monitoring the bone accumulation of 153Gd3+, 153Gd-LDTPA-protein shows a higher in vivo stability than 153Gd-DTPA-protein, the radiolabeled protein conjugate formed by the reaction of DTPA dianhydride with proteins.

Animals↗
PubMed1999

Physico-chemical stability of docetaxel premix solution and docetaxel infusion solutions in PVC bags and polyolefine containers.

We assessed the physical and chemical stability of docetaxel infusion solutions. Stability of the antineoplastic drug was determined 1.) after reconstitution of the injection concentrate and 2.) after further dilution in two commonly used vehicle-solutions, 0.9% sodium chloride and 5% dextrose, in PVC bags and polyolefine containers. Chemical stability was measured by using a stability-indicating HPLC assay with ultraviolet detection. Physical stability was determined by visual inspection. The stability tests revealed that reconstituted docetaxel solutions (= premix solutions) are physico-chemically stable (at a level > or = 95% docetaxel) for a minimum of four weeks, independent of the storage temperature (refrigerated, room temperature). Diluted infusion solutions (docetaxel concentration 0.3 mg/ml and 0.9 mg/ml), with either vehicle-solution, proved physico-chemically stable (at a level > or = 95% docetaxel) for a minimum of four weeks, when prepared in polyolefine containers and stored at room temperature. However, diluted infusion solutions exhibited limited physical stability in PVC bags, because docetaxel precipitation occurred irregularly, though not before day 5 of storage. In addition, time-dependent DEHP-teaching from PVC infusion bags by docetaxel infusion solutions must be considered.

Antineoplastic Agents, Phytogenic↗
PubMed1995

Directed evolution of a subtilisin with calcium-independent stability.

Extracellular proteases of the subtilisin-class depend upon calcium for stability. Calcium binding stabilizes these proteins in natural extracellular environments, but is an Achilles' heel in industrial environments which contain high concentrations of metal chelators. Here we direct the evolution of calcium-independent stability in subtilisin BPN'. By deleting the calcium binding loop from subtilisin, we initially destabilize the protein but create the potential to use new structural solutions for stabilization. Analysis of the structure and stability of the loop-deleted prototype followed by directed mutagenesis and selection for increased stability resulted in a subtilisin mutant with native-like proteolytic activity but 1000-times greater stability in strongly chelating conditions.

Biological Evolution↗
PubMed2002

Synthesis and properties of adducts of a hindered amine and 2-hydroxyphenylbenzotriazole as novel polymer stabilizers.

The synthesis of new types of stabilizers (a combination of 2,2,6,6-tetramethylpiperidine and 2-hydroxyphenylbenzotriazole in one molecule) is reported. Six polymerizable combined stabilizers as well as two unsaturated triazinyl-2,2,6,6-tetramethylpiperidines and three unsaturated triazinyl-2-hydroxyphenylbenzotriazoles as individual stabilizers were synthesized. Their copolymers and the terpolymers of the individual stabilizers with methyl methacrylate were obtained. Chemical bonding of the stabilizers in the polymer was confirmed spectrophotometrically. The influence of these additives on the photostability of the copolymers was studied. The participation of the combined stabilizers in the polymerization did not affect considerably the rate of copolymerization, the molecular weight and polydispersity of the copolymers. A significant stabilizing effect against photodegradation was determined.

Drug Stability↗
PubMed1980

Effect of the cholesterol content of small unilamellar liposomes on their stability in vivo and in vitro.

Small unilamellar neutral, negatively and positively charged liposomes composed of egg phosphatidylcholine, various amounts of cholesterol and, when appropriate, phosphatidic acid or stearylamine and containing 6-carboxyfluorescein were injected into mice, incubated with mouse whole blood, plasma or serum or stored at 4 degrees C. Liposomal stability, i.e. the extent to which 6-carboxyfluorescein is retained by liposomes, was dependent on their cholesterol content. (1) Cholesterol-rich (egg phosphatidylcholine/cholesterol, 7:7 molar ratio) liposomes, regardless of surface charge, remained stable in the blood of intravenously injected animals for up to at least 400min. In addition, stability of cholesterol-rich liposomes was largely maintained in vitro in the presence of whole blood, plasma or serum for at least 90min. (2) Cholesterol-poor (egg phosphatidylcholine/cholesterol, 7:2 molar ratio) or cholesterol-free (egg phosphatidylcholine) liposomes lost very rapidly (at most within 2min) much of their stability after intravenous injection or upon contact with whole blood, plasma or serum. Whole blood and to some extent plasma were less detrimental to stability than was serum. (3) After intraperitoneal injection, neutral cholesterol-rich liposomes survived in the peritoneal cavity to enter the blood circulation in their intact form. Liposomes injected intramuscularly also entered the circulation, although with somewhat diminished stability. (4) Stability of neutral and negatively charged cholesterol-rich liposomes stored at 4 degrees C was maintained for several days, and by 53 days it had declined only moderately. Stored liposomes retained their unilamellar structure and their ability to remain stable in the blood after intravenous injection. (5) Control of liposomal stability by adjusting their cholesterol content may help in the design of liposomes for effective use in biological systems in vivo and in vitro.

Animals↗
PubMed2003

Stability of extemporaneously prepared saquinavir formulations.

AIMS: The effects of pH and excipients on the stability of saquinavir in extemporaneously prepared suspensions were assessed. The stability of a developed extemporaneously prepared saquinavir suspension was then determined at 5 and 25 degrees C over 30 days. METHOD: Extemporaneous saquinavir 2 mg/mL formulations were prepared from soft gelatin capsule (Fortovase). Four batches of the formulations were buffered at pHs 2, 4, 5 and 7, whereas the other five batches were prepared in Milli-Q water, 0.5% (w/v) citric acid, 0.1% (w/v) sodium ascorbate, 10% (v/v) syrup and in vehicle containing both 0.5% (w/v) citric acid and 0.1% (w/v) sodium ascorbate. The stability of these formulations was tested at 25 degrees C. A final formulation of saquinavir suspension (60 mg/mL) containing both 10% (v/v) syrup and 0.5% (w/v) citric acid was developed and tested for stability at 5 and 25 degrees C for up to 30 days using a stability-indicating high-performance liquid chromatographic method. RESULTS: Saquinavir was most stable at pH 2-4. Formulations containing sodium ascorbate, citric acid and syrup or both citric acid and sodium ascorbate were significantly more stable than the control formulation (saquinavir 2 mg/mL in Milli-Q water). CONCLUSION: The pH for optimal stability of saquinavir was around 2-4. Besides pH adjustment, saquinavir could also be stabilized by adding anti-oxidants. The saquinavir 60 mg/mL formulation prepared with about a pH of about 4 was stable at both 5 and 25 degrees C for at least 30 days.

Capsules↗
PubMed2002

The molecular surface of proteolytic enzymes has an important role in stability of the enzymatic activity in extraordinary environments.

It is scientifically and industrially important to clarify the stabilizing mechanism of proteases in extraordinary environments. We used subtilisins ALP I and Sendai as models to study the mechanism. Subtilisin ALP I is extremely sensitive to highly alkaline conditions, even though the enzyme is produced by alkalophilic Bacillus, whereas subtilisin Sendai from alkalophilic Bacillus is stable under conditions of high alkalinity. We constructed mutant subtilisin ALP I enzymes by mutating the amino acid residues specific for subtilisin ALP I to the residues at the corresponding positions of amino acid sequence alignment of alkaline subtilisin Sendai. We observed that the two mutations in the C-terminal region were most effective for improving stability against surfactants and heat as well as high alkalinity. We predicted that the mutated residues are located on the surface of the enzyme structures and, on thebasis of three-dimensional modelling, that they are involved in stabilizing the conformation of the C-terminal region. As proteolytic enzymes frequently become inactive due to autocatalysis, stability of these enzymes in an extraordinary environment would depend on the conformational stability of the molecular surface concealing scissile peptide bonds. It appeared that the stabilization of the molecular surface structure was effective to improve the stability of the proteolytic enzymes.

Amino Acid Sequence↗
PubMed1988

Disulfide bonds and thermal stability in T4 lysozyme.

Disulfide bonds are thought to serve a stabilizing role in extracellular globular proteins, but little is known about the modes of stabilization or their mechanisms. Thermodynamic data presented here demonstrate that an engineered 3-97 disulfide bond previously shown to stabilize T4 lysozyme in vitro against irreversible thermal inactivation also stabilizes the molecule against reversible thermal unfolding. In this paper, we explore the relationship between the disulfide's thermodynamic contribution to protein folding and its role in providing resistance to irreversible thermal inactivation. In T4 lysozyme (C54V/C97S), a non-crosslinked mutant lacking the two cysteines found in the wild type, sensitivity toward irreversible thermal inactivation increases dramatically at temperatures above the melting temperature of the molecule. In addition, most of the lost activity can be restored by denaturation/renaturation with guanidine hydrochloride. In contrast, the crosslinked mutant T4 lysozyme (13C-97C/C54V) inactivates relatively slowly, even above its melting temperature, and the lost activity is not restored by denaturation/renaturation. These observations suggest that the predominant inactivation pathways for non-crosslinked T4 lysozymes are conformation related, while those for the crosslinked variant are insensitive to the conformational route and thus are susceptible only to slower processes of a chemical nature. We also show that multiple mutants, constructed to contain the 3-97 disulfide plus a temperature-sensitive lesion, are more stable than the wild type to irreversible inactivation even though they are less stable to reversible thermal unfolding. These findings together suggest that the 3-97 disulfide provides stability to irreversible inactivation primarily via a pathway that is independent of its thermodynamic contribution. The 3-97 disulfide may stabilize T4 lysozyme by restricting the unfolded state to a class of more compact structures with less exposed hydrophobic surface, compared to the unfolded states of non-crosslinked T4 lysozymes. The results have implications both for the use of the stabilizing potential of disulfide bonds in protein engineering and for their roles in protein function and evolution.

Disulfides↗